Dendrimers are polymers of spherical or other three-dimensional shapes that have precisely defined compositions and that possess a precisely defined molecular weight. Dendrimers can be synthesized as water-soluble macromolecules through appropriate selection of internal and external moieties. See, U.S. Pat. Nos. 4,507,466 and 4,568,737, incorporated by reference herein. The first well-defined, symmetrical, dendrimer family were the polyamidoamine (PAMAM) dendrimers, which are manufactured by the Dow Chemical Company. Since the synthesis and characterization of the first dendrimers, a large array of dendrimers of diverse sizes and compositions has been prepared. See, for example, Newkome, G R, Moorefield, C N and Voegtle, F. “Dendritic Molecules” VCH, Weinheim, 1996; and Liu M. and Frechet J. M. J., Pharm. Sci. Tech. Today 2(11):393 (1999).
Dendritic macromolecules are characterized by a highly branched, layered structure with a multitude of chain ends. Dendrimers are particularly well defined with a very regular and almost size monodisperse structure, while hyperbranched polymers are less well defined and have a broader polydispersity. Dendritic macromolecules are usually constructed from ABx monomers. Hyperbranched polymers are generally obtained via a polymerization reaction that generally takes place in a single series of propagation steps. Dendrimers are generally obtained by multistep iterative syntheses using either a divergent (Tomalia et al, U.S. Pat. Nos. 4,435,548; 4,507,466, 4,558,120; 4,568,737; 5,338,532) or a convergent growth approach (Hawker et al., U.S. Pat. No. 5,041,516).
Dendrimers have been conjugated with various pharmaceutical materials as well as with various targeting molecules that may function to direct the conjugates to selected body locations for diagnostic or therapeutic applications. See, for example, WO 8801178, incorporated by reference herein, see also a review by Liu M. and Frechet J. M. J., Pharm. Sci. Tech. Today 2(11):393 (1999) Dendrimers have been used to covalently couple synthetic porphyrins (e.g., hemes, chlorophyll) to antibody molecules as a means for increasing the specific activity of radiolabeled antibodies for tumor therapy and diagnosis. Roberts et al., Bioconjug. Chemistry 1:305–308 (1990); Tomalia et al., U.S. Pat. No. 5,714,166.
Rehnberg et al. (WO 99/00439) describe a method of preparing polyester dendrimers from an acetal protected 1,3-diol carboxylic acid monomer. The disclosed method relies on a carbodiimide mediated coupling of the monomer to a hydroxyl-containing initiator molecule. Ester formation via carbodiimide coupling of the monomer to the initiator molecule produces N-acyl urea-containing side products as well as the desired dendrimer. In addition the reaction with dicyclohexyl carbodiimide produces a very large amount of dicyclohexyl urea which is very difficult to remove from the desired dendrimer product. The formation of the side products is particularly problematic for the multistep synthesis of a polymeric species such as a dendrimer; the N-acyl urea side product formed in a first step reacts in a subsequent step adding a new generation to both the desired dendrimer core and to the side product. The similarity in properties between the side product and the desired dendrimer makes separation of the species difficult, and economically unfeasible for the large-scale preparation of dendrimers. As dendrimers are under intense evaluation as delivery vehicles for therapeutic and diagnostic agents, the presence of the N-acyl urea side products as well as any dicyclohexylurea impurity is seen as a serious impediment to understanding the pharmacokinetics and pharmacodynamics of these important agents, and will surely hamper their progress towards regulatory approval.
In view of the above, a method of preparing polyester dendrimers and related dendritic polymers free of urea side products, and conjugates of these dendrimers with diagnostic, therapeutic and analytic agents would represent a significant advance in the development of dendrimer-based pharmaceutical and analytical agents. Surprisingly, the present invention provides such a method and compounds produced by the method.